Plant stresses caused by a wide variety of factors including disease, environment, and storage of potato tubers (Solanum tuberosum) represent major determinants of tuber quality. Dormancy periods between harvesting and sprouting are critical to maintaining quality potatoes. Processing potatoes are usually stored between 7 and 12.degree. C. Cold storage at 2 to 6.degree. C., versus storage at 7 to 12.degree. C., provides the greatest longevity by reducing respiration, moisture loss, microbial infection, heating costs, and the need for chemical sprout inhibitors (Burton, 1989). However, low temperatures lead to cold-induced sweetening, and the resulting high sugar levels contribute to an unacceptable brown or black color in the fried product (Coffin et al., 1987, Weaver et al., 1978). The sugars that accumulate are predominantly glucose, fructose, and sucrose. It is primarily the glucose and fructose (reducing sugars) that react with free amino groups when heated during the various cooking processes such as frying via the Maillard reaction, resulting in the formation of brown pigments (Burton, 1989, Shallenberger et al., 1959). Sucrose produces a black colouration when fried due to caramelization and charring. The ideal reducing sugar content is generally accepted to be 0.1% of tuber fresh weight with 0.33% as the upper limit and higher levels of reducing sugars are sufficient to cause the formation of brown and black pigments that results in an unacceptable fried product (Davies and Viola, 1992). Although the accumulation of reducing sugars can be slowed in higher temperature (7 to 12.degree. C.) storage, this increases microbial infection and the need to use sprout inhibitors. Given the negative environmental and health risks associated with chemical use, development of pathogens resistant to pesticides, and the fact that use of current sprout inhibitors may soon be prohibited, a need exists for potato varieties that can withstand stress and long-term cold storage without the use of chemicals, without the accumulation of reducing sugars, and with greater retention of starch.
Carbohydrate metabolism is a complex process in plant cells. Manipulation of a number of different enzymatic processes may potentially affect the accumulation of reducing sugars during cold storage. For example, inhibition of starch breakdown would reduce the buildup of free sugar. Other methods may also serve to enhance the cold storage properties of potatoes through reduction of sugar content, including the resynthesis of starch using reducing sugars, removal of sugars through glycolysis and respiration, or conversion of sugars into other forms that would not participate in the Maillard reaction. However, many of the enzymatic processes are reversible, and the role of most of the enzymes involved in carbohydrate metabolism is poorly understood. The challenge remains to identify an enzyme that will deliver the desired result, achieve function at low temperatures, and still retain the product qualities desired by producers, processors, and consumers.
It has been suggested that phosphofructokinase (PFK) has an important role in the cold-induced sweetening process (Kruger and Hammond, 1988, ap Rees et al., 1988, Dixon et al., 1981, Claassen et al., 1991). ap Reese et al. (1988) suggested that cold treatment had a disproportionate effect on different pathways in carbohydrate metabolism in that glycolysis was more severely reduced due to the cold-sensitivity of PFK. The reduction in PFK activity would then lead to an increased availability of hexose-phosphates for sucrose production. It was disclosed in European Patent 0438904 (Burrell et al., Jul. 31, 1991) that increasing PFK activity reduces sugar accumulation during storage by removing hexoses through glycolysis and further metabolism. A PFK enzyme from E. coli was expressed in potato tubers and the report claimed to increase PFK activity and to reduce sucrose content in tubers assayed at harvest. However it has been shown that pyrophosphate:fructose 6-phosphate phosphotransferase (PFP) remains active at low temperatures (Claassen et al., 1991). PFP activity can supply fructose 6-phosphate for glycolysis just as PFK can, since the two enzymes catalyse the same reaction. Therefore, the efficacy of this strategy for improving cold storage quality of potato tubers remains in doubt. Furthermore, removal of sugars through glycolysis and further metabolism would not be a preferred method of enhancing storage properties of potato tubers because of the resultant loss of valuable dry matter through respiration.
It has also been suggested that ADPglucose pyrophosphorylase (ADPGPP) has an important role in the cold-induced sweetening process. It was disclosed in International Application WO 94/28149 (Barry, et al., filed May 18, 1994) that increasing ADPGPP activity reduces sugar accumulation during storage by re-synthesising starch using reducing sugars. An ADPGPP enzyme from E. coli was expressed in potato tubers under the control of a cold-induced promoter and the report claimed to increase ADPGPP activity and lower reducing sugar content in tubers assayed at harvest and after cold temperature storage. However, this strategy does not eliminate starch catabolism but instead increases the rate of starch resynthesis. Thus, catabolism of sugars through glycolysis and respiration occurs and re-incorporation into starch is limited. Up regulation of ADPGPP would not be a preferred method of enhancing storage properties of potato tubers because of the resultant loss of valuable dry matter through respiration. Again, a method involving the reduction of catabolism of starch would be preferable as dry matter would be retained.
The degradation of starch is believed to involve several enzymes including .alpha.-amylase (endoamylase), .beta.-amylase (exoamylase), amyloglucosidase, and .alpha.-glucan phosphorylase (starch phosphorylase). By slowing starch catabolism, accumulation of reducing sugars should be prevented and the removal of sugars through glycolysis and further metabolism would be minimized.
Three different isozymes of .alpha. glucan phosphorylase have been described. The tuber L-type .alpha.1,4 glucan phosphorylase (EC 2.4.1.1) isozyme (GLTP) (Nakano and Fukui, 1986) has a low affinity for highly branched glucans, such as glycogen, and is localized in amyoplasts. The monomer consists of 916 amino acids and sequence comparisons with phosphorylases from rabbit muscle and Escherichia coli revealed a high level of homology, 51% and 40% amino acids, respectively. The nucleotide sequence of the GLTP gene and the amino acid sequence of the GLTP enzyme are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The H-type tuber .alpha.-glucan phosphorylase isozyme H (GHTP) (Mori et al., 1991) has a high affinity for glycogen and is localized in the cytoplasm. The gene encodes for 838 amino acids and shows 63% sequence homology with the tuber L-type phosphorylase but lacks the 78-residue insertion and 50-residue amino-terminal extension found in the L-type polypeptide. The nucleotide sequence of the GHTP gene and the amino acid sequence of the GHTP enzyme are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. A third isozyme has been reported (Sonnewald et al., 1995) that consists of 974 amino acids and is highly homologous to the tuber L-type phosphorylase with 81% identity over most of the polypeptide. However, the regions containing the transit peptide and insertion sequence are highly diverse. This isozyme is referred to as the leaf L-type phosphorylase since the mRNA accumulates equally in leaf and tuber, whereas the mRNA of the tuber L-type phosphorylase accumulates strongly in potato tubers and only weakly in leaf tissues. The tuber L-type phosphorylase is mainly present in the tubers and the leaf L-type phosphorylase is more abundunt in the leaves (Sonnewald et al., 1995). The nucleotide sequence of the leaf L-type phosphorylase gene and the amino acid sequence of the leaf L-type phosphorylase enzyme are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
The role of the various starch degrading enzymes is not clear, however, and considerable debate has occurred over conflicting results. For example, reduced expression of the leaf L-type phosphorylase (Sonnewald et al., 1995) had no significant influence on starch accumulation. Sonnewald et al. (1995) reported that constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of .alpha. glucan phosphorylase L-type activity in leaf tissue, but had no effect in potato tuber tissue. Since the antisense repression of the .alpha. glucan phosphorylase activity had no significant influence on starch accumulation in leaves of transgenic potato plants, the authors concluded that starch breakdown was not catalysed by phosphorylases. Considering the high level of sequence homology between identified .alpha. glucan phosphorylase isozymes, a similar negative response would be expected with the H-type (GHTP) and L-type tuber (GLTP) isozymes.
In view of the foregoing, there remains a need for potato plants which produce tubers exhibiting reduced conversion of starches to sugars during propagation and during storage at ambient and reduced temperatures, particularly at temperatures below 7.degree. C.